Systems and Methods for Producing Gastrointestinal Tissues

ABSTRACT

Aspects of the disclosure relate methods and synthetic scaffolds for regenerating gastrointestinal tissue (e.g., esophageal tissue).

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional application of U.S. Ser. No. 15/350,970currently pending which claims priority to and the benefit of U.S.Provisional Patent Application Ser. No. 62/254,700 filed Nov. 12, 2015and U.S. Provisional Patent Application Ser. No. 62/276,715 filed Jan.8, 2016, the entire disclosure of which are both hereby incorporated byreference.

TECHNICAL FIELD

This disclosure relates to engineered tissues that are useful forreplacement or repair of damage tissues.

BACKGROUND

Engineered biological tissues that are useful for replacement or repairof damaged tissues are often produced by seeding cells on syntheticscaffolds and exposing the cells to conditions that permit them tosynthesize and secrete extracellular matrix components on the scaffold.Different techniques have been used for producing synthetic scaffolds,including nanofiber assembly, casting, printing, physical spraying(e.g., using pumps and syringes), electrospinning, electrospraying andother techniques for depositing one or more natural or syntheticpolymers or fibers to form a scaffold having a suitable shape and sizefor transplanting into a subject (e.g., a human subject, for example, inneed of an organ or region of engineered tissue).

It is estimated that over 500,000 individuals worldwide are diagnosedwith esophageal malignancy each year. Congenital malformations of theesophagus, such as esophageal atresia, have an average prevalence of2.44 per 10,000 births. Chronic esophageal stricture after esophagealinjury is also common. While there have been advances in minimization ofthe extent of esophageal resection for early stage malignant disease,such as endoscopic mucosal resection, the mainstay of treatment for manyesophageal disorders is surgical esophagectomy. Traditionally,autologous conduits such as stomach, small bowel, or colon are harvestedand rerouted into the chest to restore gastrointestinal continuity. Manychildren with esophageal atresia or patients affected by either traumaor caustic injury to the esophagus ultimately undergo similarreconstruction. These treatment modalities are associated with highmorbidity and mortality.

Autologous conduits are traditionally used because of the complexstructure of the esophagus. Comprised of stratified squamous epithelium,submucosa and outer circular and longitudinal muscle layers, thesemultiple layers of the esophagus provide a barrier to contain oralintake and contamination from escape outside of the gastrointestinaltract. Furthermore, the combined layers provide a physiologicalmechanism for propulsion, and management of stresses during passage ofthe bolus either during swallowing or emesis.

It would be desirable to provide structure as well as a method of makinga structure that can support tissue regeneration.

SUMMARY

Disclosed herein are implementations that pertain to synthetic scaffoldsand related systems that enable production of gastrointestinal tissues(e.g., tissues of the esophagus, stomach, intestine, colon, or otherhollow gastrointestinal tissue). In some embodiments, scaffolds provideguides for gastrointestinal (e.g., esophageal) tissue growth andregeneration in a subject. In some embodiments, the regeneratedgastrointestinal tissue comprises muscle tissue, nervous system tissue,or muscle tissue and nervous system tissue. In some embodiments,gastrointestinal (e.g., esophageal) tissue is regenerated surrounding ascaffold. In some embodiments, the scaffold is not incorporated into thefinal regenerated tissue (e.g., the new esophageal tissue does notincorporate the scaffold into the regenerated esophageal walls).Accordingly, aspects of the disclosure relate to guided tissueregeneration where a scaffold provides support and/or signals thatpromote host tissue regeneration without the scaffold needing to beincorporated into the regenerated tissue (e.g., without the scaffoldproviding structural or functional support in the final regeneratedtissue).

In some embodiments, a gastrointestinal (e.g., esophageal) scaffoldincludes biodegradable and/or bioresorbable material that is resorbedafter gastrointestinal (e.g., esophageal) tissue regeneration isinitiated (e.g., after functional esophageal tissue is regenerated).

In some embodiments, a gastrointestinal (e.g., esophageal) scaffoldincludes one or more structures that can be used to assist in removingthe scaffold after gastrointestinal (e.g., esophageal) tissueregeneration is initiated (e.g., after functional esophageal tissue isregenerated).

In some embodiments, a scaffold is cellularized with one or more celltypes prior to implantation. In some embodiments, the cells areautologous cells. In some embodiments, the cells are progenitor or stemscells. In some embodiments, the cells are obtained from bone marrow,adipogenic tissue, esophageal tissue, or other suitable tissue. In someembodiments, the cells can be obtained from various allogenic sources,including but not limited to sources such as amniotic fluid, cord boldand the like. In some embodiments, the cells are mesenchymal stem cells(MSCs)

In some embodiments, a scaffold is implanted at a site that provides asufficient stem cell niche (e.g., an esophageal or othergastrointestinal site that provides a stem cell niche) for regeneratingtissue in the subject. In some embodiments, without wishing to be boundby theory, the scaffold and/or cells that are provided on the scaffoldhelp promote growth and/or regeneration of gastrointestinal tissue fromhost stem cells present at the site of scaffold implantation.

In some aspects, the disclosure relates to the discovery that growth ofesophageal tissues can be promoted or enhanced by the presence ofsynthetic scaffolds that are engineered to replace or repair naturalstructural patterns and/or functional properties of diseased or injuredtissues or organs, without the scaffolds becoming fully integrated intothe final regenerated tissue. Thus, in some aspects, the disclosureprovides a method for promoting or enhancing growth of gastrointestinal(e.g., esophageal) tissue, the method comprising: delivering to agastrointestinal (e.g., esophageal) region of a subject a syntheticscaffold, wherein delivery of the synthetic scaffold results in growthof new gastrointestinal (e.g., esophageal) tissue in that region of thesubject. In some embodiments, the diseased or injured gastrointestinalregion is removed (e.g., surgically) prior to implanting the scaffold.In some embodiments, the scaffold is an approximately tubular structurethat is implanted (e.g., sutured to the ends of the remaininggastrointestinal tissue after removal of the diseased or damagedtissue). In some embodiments, the implanted scaffold is shorter than thetissue that was removed (e.g., 5-50% shorter). In some embodiments, theremaining gastrointestinal tissue near the site of the implant isstretched when the tissue is attached (e.g., sutured) to the both endsof the scaffold. In some embodiments, new gastrointestinal (e.g.,esophageal) tissue is regenerated over the implanted scaffold withoutbeing fully integrated with the scaffold. In some embodiments, the wallsof the regenerated tissue do not incorporate the walls of the scaffoldeven though the scaffold can be retained within the lumen of theregenerated tissue. In some embodiments, the scaffold can be removedfrom the lumen formed by the regenerated tissue at a suitable point inthe tissue regeneration process.

In some embodiments, the growth of new gastrointestinal (e.g.,esophageal) tissue results in the formation of functional tissue (e.g.,a functional esophagus) that does not require the continued presence ofthe scaffold for function.

In some embodiments, the synthetic scaffold is resorbable or dissolvableunder physiological conditions. In some embodiments, the syntheticscaffold is removed from the gastrointestinal (e.g., esophageal) regionof the subject after the formation of a functional esophagus.

In some embodiments, methods and compositions described herein also canbe used for tracheal and/or bronchial tissue regeneration.

These and other aspects are described in more detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure is best understood from the following detaileddescription when read in conjunction with the accompanying drawings. Itis emphasized that, according to common practice, the various featuresof the drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.

FIG. 1A is a perspective view of an embodiment of a synthetic scaffoldas disclosed herein with a portion being rendered in partialcross-section;

FIG. 1B is a photomicrograph of a surface of a tubing surface of anembodiment of the synthetic scaffold as disclosed herein;

FIG. 1C side perspective view of a second embodiment of a syntheticscaffold as disclosed herein;.

FIG. 2 is a non-limiting depiction of the biological layers of anesophagus; and

FIG. 3 illustrates a non-limiting example of regenerated esophagealtissue in comparison with corresponding native tissue;

FIG. 4A is a SEM photomicrograph of an outer surface region of anembodiment of the synthetic scaffold as disclosed herein showingcellular growth after seven days of bioreaction taken at 5000×;

FIG. 4B is a photomicrograph of an outer surface region of an embodimentof the synthetic scaffold as disclosed herein showing cellular growthafter seven days of bioreaction;

FIG. 5 is a process diagram of an embodiment an embodiment of theregeneration method as disclosed herein;

FIG. 6 is an overall study flow for an embodiment of the process asdisclosed herein including generation of a cellularized scaffold andsubsequent implantation

FIG. 7A is are SEMs of samples of an electrospun scaffold according toan embodiment as disclosed herein taken at 1000×, 2000× and 5000×respecitvely;

FIG. 7B is a graphic depiction of representative uniaxial mechanicaltesting loading of pre-implantation and post-implantation electrospunscaffolds according to an embodiment as disclosed herein;

FIG. 7C is a Table directed to the uniaxial mechanical properties ofpre- and post-implantations scaffolds prepared according to anembodiment as disclosed herein;

FIG. 8 is a diagrammatic representation of flow cytometry of MSCsisolated and propagated from adipose tissue for up to 5 passages;

FIG. 9 is an overview of implantation surgery according to an embodimentas disclosed herein;

FIG. 10 is a representation of a timeline according to an embodiment ofthe process as disclosed herein;

FIG. 11A is a photograph of regenerated esophageal tissue that islocated on the interior of the esophagus of a first test subject anesophageal resection site of the first test subject at defined intervalstaken after removal of an embodiment of the scaffold device as disclosedherein at 3 to 4 weeks post-surgery;

FIG. 11B is a photograph of regenerated tissue that is located on theinterior of the esophagus at the esophageal resection site of FIG. 11Aat an intermediate interval after removal of the scaffold device showingtissue growth;

FIG. 11C is a photograph of regenerated tissue that is located on theinterior of the esophagus at the esophageal resection site of FIG. 11Aat an interval subsequent to the intermediate interval of FIG. 11Bshowing tissue growth;

FIGS. 12A is a photograph of regenerated esophageal tissue that islocated on the interior of the esophagus at an esophageal resection siteof a second test subject taken after removal of an embodiment of thescaffold device as disclosed herein at 3 to 4 weeks;

FIG. 12B is a photograph of regenerated tissue that is located on theinterior of the esophagus at the esophageal resection site of FIG. 12Aat an intermediate interval after removal of the scaffold device showingtissue growth;

FIG. 12C is a photograph of regenerated tissue that is located on theinterior of the esophagus at the esophageal resection site of FIG. 12Aat an intermediate interval after removal of the scaffold device showingtissue growth subsequent to the tissue growth depicted in FIG. 12B;

FIG. 12D is a photograph of regenerated tissue that is located on theinterior of the esophagus at the esophageal resection site of FIG. 12Aat an intermediate interval after removal of the scaffold device showingtissue growth subsequent to the tissue growth depicted in FIG. 12C;

FIG. 12E is a photograph of regenerated tissue that is located on theinterior of the esophagus at the esophageal resection site of FIG. 12Aat an intermediate interval after removal of the scaffold device showingtissue growth subsequent to the tissue growth depicted in FIG. 12D;

FIG. 13A is a photograph of tissues from a representative test animalesophagus at 2.5 months post implantation including the surgical siteand adjacent distal and proximal tissues excised for histologicalanalysis;

FIG. 13B is a photograph of a magnified cross-sectional sample of mucosatissue taken from proximal section 1 of FIG. 13A;

FIG. 13C is a photograph of a magnified cross-sectional sample of mucosatissue taken from proximal section 2 of FIG. 13A;

FIG. 13D is a photograph of a magnified cross-sectional sample ofsubmucosa tissue taken from proximal section 1 of FIG. 13A;

FIG. 13E is a photograph of a magnified cross-sectional sample ofsubmucosa tissue taken from proximal section 2 of FIG. 13A;

FIG. 13F is a photograph of a magnified cross-sectional sample of mucosatissue taken from distal section 3 of FIG. 13A;

FIG. 13G is a photograph of a magnified cross-sectional sample of mucosatissue taken from distal section 4 of FIG. 13A;

FIG. 13H is a photograph of a magnified cross-sectional sample of mucosatissue taken from distal section 4 of FIG. 13A;

FIG. 131 is a photograph of a magnified cross-sectional sample ofsubmucosa tissue taken from distal section 4 of FIG. 13A;

FIG. 14A is a photograph of tissues of pig esophagus for histologicalanalysis at 2.5 months post implantation with an embodiment of thescaffold as disclosed herein;

FIG. 14B is photograph of a magnified cross-sectional sample taken asection B of FIG. 14A illustrating the presence of mucosal tissue;

FIG. 14C is photograph of a magnified cross-sectional sample taken asection C of FIG. 14A illustrating the presence of mucosal tissue;

FIG. 14D is photograph of a magnified cross-sectional sample taken asection D of FIG. 14A illustrating the presence of mucosal and submucosatissue and muscular layers;

FIG. 14E is photograph of a magnified cross-sectional sample taken asection E of FIG. 14A illustrating the presence of mucosal and submucosatissue and muscular layers;

FIG. 14F is a photograph of a cross-sectional sample of esophagealtissue of FIG. 14A used for Ki67 immunoreactivity analysis;

FIG. 14G is a photograph of a cross-sectional sample of esophagealtissue of FIG. 14A used for CD31 immunoreactivity analysis;

FIG. 14H is a photograph of a cross-sectional sample of esophagealtissue of FIG. 14A used for CD3c immunoreactivity analysis;

FIG. 14I is a photograph of a cross-sectional sample of esophagealtissue of FIG. 14A used for aSMA immunoreactivity analysis;

FIG. 14J is a photograph of a cross-sectional sample of esophagealtissue of FIG. 14A used for Transgelin/SMA22a immunoreactivity analysis;and

FIG. 14K is a photograph of a cross-sectional sample of esophagealtissue of FIG. 14A.

DETAILED DESCRIPTION

Aspects of the disclosure relate in part to the remarkable discoverythat inserting a synthetic scaffold into the esophageal region of asubject can promote or enhance the regeneration of new esophageal tissue(e.g., a complete and functional esophagus) in the subject without fullyincorporating the scaffold into the regenerated tissue. Thus, in someembodiments, the disclosure provides a method for promoting or enhancinggrowth of gastrointestinal (e.g., esophageal) tissue, the methodcomprising: delivering to the gastrointestinal (e.g., esophageal) regionof a subject a synthetic scaffold, wherein delivery of the syntheticscaffold results in growth of new gastrointestinal (e.g., esophageal)tissue in that region of the subject.

Tissue that is regenerated using methods described herein can be anygastrointestinal tissue, such as tissues of the esophagus, stomach,intestine, colon, rectum, or other hollow gastrointestinal tissue. Insome aspects, the disclosure is based, in part, on the surprisingdiscovery that methods described herein result in the regeneration ofgastrointestinal tissue comprising muscle tissue, nervous system tissue,or muscle tissue and nervous system tissue.

In some embodiments, the synthetic scaffold is resorbable or dissolvableunder physiological conditions (e.g., within a time period correspondingapproximately to the time required for tissue regeneration). In someembodiments, at least a portion of the synthetic scaffold is resorbableor dissolvable under suitable physiological conditions.

In some embodiments, the synthetic scaffold is removed from the subjectafter the formation of a regenerated functional tissue (e.g., esophagusor portion thereof).

In some embodiments, a scaffold is designed to be readily retrievable byhaving a) one or more reversible attachments that can be easier toremove than a suture, for example to help disconnect the scaffold fromthe surrounding tissue (e.g., esophagus) after tissue regeneration,and/or b) one or more features that can be used to help retrieve thescaffold, for example after it has been disconnected from thesurrounding tissue (e.g., adjacent esophageal tissue).

Non-limiting examples of reversible attachments include mechanicalmechanisms (for example hooks and loops, connectors such as stents, orother mechanical attachments that can be disconnected) and/or chemicalmechanisms (for example biodegradable or absorbable attachments and/orattachments that can be selectively removed by chemical or enzymaticmeans). In some embodiments, absorbable staples can be used. In someembodiments, absorbable staples comprise a co-polymer ofpolylactide-polyglycolide for example, or any other absorbable blend ofmaterial.

In some embodiments, surgical implantation and/or retrieval of ascaffold can be performed with thoracoscopic assistance.

Non-limiting examples of structural features that can assist in theretrieval or removal of a scaffold (e.g., after it is disconnected fromthe surrounding gastrointestinal tissue) include holes, indents,protrusions, or other structural features, or any combination thereofthese structural features are located only on the outer surface of thescaffold. One or more of these structural features can be used to helpgrip or hold a tool (e.g. a grasper) that is being used to retrieve thescaffold. In some embodiments, one or more of these structural featurescan be located at only one end of the scaffold (e.g., the end that isproximal to the mouth of the subject). In some embodiments, one or moreof these structural features can be located at both ends, or throughoutthe length of the scaffold. In some embodiments, one or more of thesestructural features are located only on the outer surface of thescaffold. In some embodiments, one or more of these structural featuresare located only on the inner surface of the scaffold. In someembodiments, one or more of these structural features are located onboth the outer and inner surfaces of the scaffold. In some embodiments,a scaffold is reinforced (e.g., is thicker and/or includes strongermaterial) at or around the location of one or more structural featuresthat are used to retrieve the scaffold.

In some embodiments, a disconnected scaffold can be removedendoscopically via the lumen of the airway leading to the esophagus. Insome embodiments, a disconnected scaffold can be removed surgically

In some embodiments, the subject has diseased (e.g., cancerous) orinjured gastrointestinal tissue that needs to be replaced. In someembodiments, the subject is a human (e.g., a human patient).

In some embodiments, the disclosure provides engineered scaffolds thatcan be used to replace or repair an esophagus or a portion thereof. Insome embodiments, esophageal scaffolds described herein may be used forpromoting tissue regeneration (e.g., a regenerated esophagus or portionthereof) to replace a tissue in a subject (e.g., a human). For example,subjects (e.g., a human) having certain cancers (e.g., esophagealcancer) may benefit from replacement of a tissue or organ affected bythe cancer. Without wising to be bound by any particular theory,synthetic scaffolds described herein promote the growth of new tissue(e.g., esophageal tissue) in a subject and therefore provide atherapeutic benefit to the subject.

In some embodiments, the growth of new esophageal tissue results in theformation of a functional esophagus in the subject. In some embodiments,the new esophageal tissue does not incorporate the scaffold into theregenerated esophageal walls. In some embodiments, the scaffold isdesigned and manufactured to be absorbable and/or readily retrievableafter the esophageal tissue has regenerated. In some embodiments, thescaffold is designed to be at least partially absorbable.

In some embodiments, a synthetic scaffold has a size and shape thatapproximates the size and shape of a diseased or injuredgastrointestinal (e.g., esophageal) region that is being replaced.

In some embodiments, a scaffold will have at least two layers. Thescaffold can have an approximately tubular structure in certainembodiments. FIG. 1 illustrates a non-limiting embodiment of a scaffold10 having an approximately tubular body 12 having an interiorly orientedsurface 14 and an exteriorly oriented surface 16. In some embodiments, alateral cross-section of the scaffold 10 is approximately circular. Insome embodiments, a lateral cross-section is approximately “D” shaped.However, scaffolds 10 having other cross-sectional shapes can be used.Scaffold 10 can have any suitable length and diameter depending on thesize of the corresponding tissue being regenerated. In some embodiments,a scaffold 10 can be from around 1-10 cms in length (for example 3-6cms, e.g., about 4 cms) in certain embodiments, or 10-20 cms long inother embodiments. However, it is contemplated that shorter or longerscaffolds 10 can be used depending on the application, needs of thepatient and/or locations in the gastrointestinal tract requiringtreatment. In some embodiments, a scaffold 10 can have an inner diameterof 0.5 to 5 cms. However, scaffolds with smaller or larger innerdiameters can be used depending on the application, needs of the patientand/or locations in the gastrointestinal tract requiring treatment.

In some embodiments, the length of scaffold 10 can be shorter than thelength of a gastrointestinal (e.g., esophageal) region being replaced.In some embodiments, the scaffold 10 has a length that is 50-95% (forexample, about 50-60%, 60-70%, 70-80%, 80-90%, about 80%, about 85%,about 90%, or about 95%) of the length of the tissue being replaced.Without being bound to any theory, it is believed that certain regionsof the associated gastrointestinal region can respond positively totraction force exerted on the associated organ tissue resulting thegeneration of certain bio-organically mediated signals that initiate orpromote tissue growth and differentiation.

In certain embodiments, the length of scaffold 10 can have a lengthlonger than the length of a gastrointestinal (e.g., esophageal) regionbeing replaced. In some embodiments, the scaffold 10 has a length thatis between 100% and 150% (for example, about 100-110%, 110-120%,120-130%, 130-140%, about 100%, about 105%, about 110%, or about 115%)of the length being replaced. It is contemplated that the length of thescaffold will be that necessary to effectively replace the effectedregion. In certain situations, it is contemplated that a scaffold 10will have a length that is longer than the replaced gastrointestinalregion to effectively position the scaffold and reduce or minimizetrauma and ischemia in the effected or associated regions.

In some embodiments, a scaffold 10 can be composed of a single layer ofsynthetic material. However, it is within the purview of this disclosurethat the scaffold 10 also can include more than one layer of syntheticmaterial.

Accordingly, in some embodiments, the synthetic scaffold 10 can becomposed of multiple layers (e.g., 2 or more layers, for example 2, 3,4, 5, or more layers). In some embodiments, one or more layers are madeof the same material. In some embodiments, the different layers are madeof different materials (e.g., different polymers and/or differentpolymer arrangements). Synthetic scaffolds 10 as disclosed herein mayinclude two or more different components that are assembled to form thescaffold as it exists e.g., prior to cellularization and/orimplantation. In some embodiments, a synthetic scaffold 10 includes twoor more layers that are brought into contact with each other, forexample by the synthetic techniques that are used to manufacture thescaffold 10. In some embodiments, a scaffold 10 may be synthesized usinga technique that involves several steps that result in two or morelayers being brought together (e.g., the application of a layer ofelectrospun material onto a portion of the scaffold that was previouslymade, such as an prior layer of electrosprayed material, a prior layerof electrospun material, a surface of a different component (e.g., abraided tube or mesh) that is being incorporated into the scaffold, or acombination of two or more thereof).

In the embodiment as depicted in FIG. 1A, scaffold 10 includes at leastone outer layer 18 that defines the outer surface 14 of the scaffoldbody 12. The scaffold 10 includes at least one additional inwardlyoriented layer 20. In the embodiment as illustrated, the at least oneadditional inwardly oriented layer 20 is in direct contact with aninwardly oriented face of the outer layer 18. Where desired or required,the at least one inwardly oriented layer 20 can be configured to providestructural support to the associated scaffold body 12. IN the embodimentdepicted, in FIG. 1A, the at least one inwardly oriented layer 20 can beconfigured as a suitable mesh or braid positioned circumferentiallyaround at least a portion of the longitudinal length of the scaffoldbody 12. In other embodiments, it is contemplated that the at least oneinwardly oriented layer 20 can be composed of a suitable polymericlayer. In the embodiment as illustrated in FIG. 1A. the body 12 ofscaffold 10 includes at least one layer 22 that is located interior tothe mesh or braid layer 20.

Where desired or required, the scaffold 10 can have a wall thicknessthat is generally uniform. However, in some embodiments, the wallthickness can vary at specific regions of the body 12. In someembodiments, the wall thickness at one or both ends 24, 26 of the body12 of scaffold 10 is different (e.g., thicker) than the walls of thecentral portion 28 of the scaffold 10 (not shown). In some embodiments,the thicker wall regions are stronger and provide greater support forsutures that are connected to one or both ends 24, 26 of the scaffold 10when the scaffold is connected to surrounding gastrointestinal tissue.The thicker wall region(s) can also include discrete configurations thatfacilitate suturing. Non-limiting examples of such configurationsinclude tubes, wholes, etc.

In certain embodiments, at least the exteriorly oriented surface 14defined on the outwardly oriented layer 18 can be composed of anelectrospun polymeric material. In certain embodiments, it iscontemplated that the outwardly oriented wall 18 can be composed ofelectrospun polymeric material. In certain embodiments, the electrospunoutwardly oriented layer can be in direct contact with a suitable braidmaterial layer 20.

Fiber Orientation

Electrospun fibers can be isotropic or anisotropic. In some embodiments,fibers in different layers can have different relative orientations. Insome embodiments, fibers in different layers can have substantially thesame orientation. Fiber orientation can be altered in each layer of acomposite or sandwich scaffold in addition.

In some embodiments, scaffolds with different porosities can be used. Insome embodiments, one or more layers of a scaffold permit substantiallycomplete cellular penetration and uniform seeding. In some embodiments,one or more layers of the scaffold may be constructed to prevent thepenetration of one or more cell types, for example by densely packingthe fibers. Controlling fiber diameter can be used to change scaffoldporosity as the porosity scales with fiber diameter. Alternatively,blends of different polymers may be electrospun together and one polymerpreferentially dissolved to increase scaffold porosity. The propertiesof the fibers can be controlled to optimize the fiber diameter, thefiber spacing or porosity, the morphology of each fiber such as theporosity of the fibers or the aspect ratio, varying the shape from roundto ribbon-like. In some embodiments, the mechanical properties of eachfiber may be controlled or optimized, for example by changing the fibercomposition, and/or the degradation rate.

In certain embodiments, the electrospun fiber material can provide acontoured surface such as a that depicted in FIG. 1B. In certainembodiments, at least one electrosupn layer in scaffold 10 can be apolymeric fiber material such as polycarbonate polyurethane and can beproduced by dissolving polycarbonate-polyurethane in a suitable solventsuch as hexa fluoroisopropanol (HFIP) that is spun and dried.

The spacing and porosity of the electrospun fiber material can be thatsuch that cells seeded on the scaffold surface can adhere in suspendedoverlying relationship between respective fibers to permit the seededcellular material to form sheets thereon as illustrated in FIG. 4A and4B.

Layering of Synthetic Scaffolds

Aspects of the disclosure relate to methods for producing syntheticscaffolds. In some embodiments, tubular synthetic scaffolds (e.g., asynthetic esophageal scaffold) are produced on a mandrel (e.g., bydepositing material via electro spraying and/or electro spinning).

In some embodiments, one or more layers of a synthetic scaffold providestructural support to the scaffold, conferring a desired mechanicalproperty to the scaffold. In some embodiments, a braided material (e.g.,a braided tube, for example a nitinol braid, a PET braid, or a braid ofother metallic or non-metallic material) can be inserted between twodifferent layers of a scaffold to provide structural support. Thecompression force of the braided material (e.g., the force that thebraid can exert on the next layer of material, for example the outerelectrospun layer of material) can be controlled by controlling the pickcount of the braid. In some embodiments, a braid can be coated (e.g., bydipping or other technique) in an organic solvent to help attach it toone or more other layers of the scaffold 10. In some embodiments, thelength of the braid 20 does not extend to the ends of the scaffold body12. In some embodiments, one or both ends of the scaffold 10 consist oftwo or more layers of material without a braided layer, whereas thecentral portion 28 of the scaffold body 12 includes an additionalbraided layer.

In some embodiments, one or more layers of a synthetic scaffold providea barrier in the scaffold, creating a separation (e.g., a relativelyimpermeable separation) between an inner space (e.g., a lumenal space)and an external space. In some embodiments, a barrier can be anelectrosprayed polyurethane (PU) layer.

In some embodiments, different layers of a scaffold 10 can include oneor more polymers (e.g., polyethylene terephthalate (PET), PU, or blendsthereof). In some embodiments, a scaffold 10 can include a nitinol braidsandwiched between an inner PU layer (e.g., that was electrosprayed orelectrospun onto a mandrel) and an outer PU layer (e.g., that waselectrosprayed onto the braided material).

In certain embodiments the scaffold 10 can be formed using a scaffoldsupport or mandrel. In some embodiments, a scaffold support or mandrelmay be coated with a material (e.g., PLGA or other polymer) prior todepositing one or more layers of PU, PET, or a combination thereof.

In certain embodiments, the material in the braid or mesh layer can becomposed of absorbable polymeric material.

Scaffold Production-Fiber Materials

In some embodiments, one or more layers of a scaffold may be constructedfrom fibrous material. In some embodiments, scaffolds comprise one ormore types of fiber (e.g., nanofibers). In some embodiments, scaffoldscomprise one or more natural fibers, one or more synthetic fibers, oneor more polymers, or any combination thereof. It should be appreciatedthat different material (e.g., different fibers) can be used in methodsand compositions described herein. In some embodiments, the material isbiocompatible so that it can support cell growth. In some embodiments,the material is permanent, semi-permanent (e.g., it persists for severalyears after implantation into the host), or rapidly degradable (e.g., itis resorbed within several weeks or months after implantation into thehost).

In some embodiments, the scaffold comprises or consists of electrospunmaterial (e.g., macro or nanofibers). In some embodiments, theelectrospun material contains or consists of PET (polyethyleneterephthalate (sometimes written poly(ethylene terephthalate)). In someembodiments, the electrospun material contains or consists ofpolyurethane (PU). In some embodiments, the electrospun materialcontains or consists of PET and PU.

In some embodiments, the artificial scaffold may consist of or includeone or more of any of the following materials: elastic polymers (e.g.,one or more polyurethanes (PU), for example polycarbonates and/orpolyesters), acrylamide polymers, Nylon, resorbable polysulfone polymersand mixtures thereof. In some embodiments, the scaffold may consist ofor include polyethylene, polypropylene, poly(vinylchloride),polymethylmethacrylate (and other acrylic resins), polystyrene, andcopolymers thereof (including ABA type block copolymers),poly(vinylidene fluoride), poly(vinylidene chloride), polyvinylalcoholin various degrees of hydrolysis (e.g., 87% to 99.5%) in cross-linkedand non-cross-linked forms. In certain embodiments, the polymericcompound can also include compounds or processes to increase thehydrophilic nature of the polymer. In certain embodiments, this caninvolve incorporating compounds such as block copolymers based onethylene oxide and propylene oxide. It is also contemplated that thehydrophilic nature of the polymer can be increase by suitable plasmatreatment if desired or required.

In some embodiments, the scaffold may consist of or include blockcopolymers. In some embodiments, addition polymers like polyvinylidenefluoride, syndiotactic polystyrene, copolymer of vinylidene fluoride andhexafluoropropylene, polyvinyl alcohol, polyvinyl acetate, amorphousaddition polymers, such as poly(acrylonitrile) and its copolymers withacrylic acid and methacrylates, polystyrene, poly(vinyl chloride) andits various copolymers, poly(methyl methacrylate) and its variouscopolymers, and PET (polyethylene terephthalate (sometimes writtenpoly(ethylene terephthalate))) can be solution spun or electrospun andcombined with any other material disclosed herein to produce a scaffold.In some embodiments, highly crystalline polymers like polyethylene andpolypropylene may be solution spun or combined with any other materialdisclosed herein to produce a scaffold.

In some embodiments, one or more polymers are modified to reduce theirhydrophobicity and/or increase their hydrophilicity after the scaffoldsynthesis, but before scaffold cellularization and/or implantation.

The electrospun fibers can have a dimeter less than 10 micometers incertain embodiments. In certain embodiments, the electrospum fibers. Incertain embodiments, the electrospun fibers can have a diameter between3 and 10 micrometers. The electrospun fibers can have a dimeter between3 and 5 micrometers in certain embodiments.

In certain embodiments, it is contemplated that the material in thebraid layer can be made in whole or in part of bioabsorbable materialssuch as PLGA and the like. it is also contemplated that, in certainconfigurations, the braid material can be loaded materials and compoundsthat can promote and/or support tissue growth and regeneration.Non-limiting examples of such compounds and materials include one ormore of the following: antibiotics, growth factors and the like.

Electrospinning

In some embodiments, scaffolds are produced that include one or morelayers (e.g., of PU and/or PET) produced via electrospinning.Electrospun material can be used for a variety of applications,including as a scaffold for tissue engineering. Appropriate methods ofelectrospinning polymers may include those described in Doshi andReneker. Electrospinning process and application of electrospun fibers.J Electrostat. 1995;35:151-60.; Reneker D H, Chun I. Nanometer diameterfibers of polymer produced by electrospinning. Nanotechnology. 1996;7:216-23; Dzenis Y. Spinning continuous fibers for nanotechnology.Science. 2004; 304:1917-19; or Vasita and Katti. Nanofibers and theirapplications in tissue engineering. Int J. Nanomedicine. 2006; 1(1):15-30, the contents of which relating to electrospinning areincorporated herein by reference. Electrospinning is a versatiletechnique that can be used to produce either randomly oriented oraligned fibers with essentially any chemistry and diameters ranging fromnm scale (e.g., around 15 nm) to micron scale (e.g., around 10 microns).

In some embodiments, electrospinning and electrospraying techniques usedherein involve using a high voltage electric field to charge a polymersolution (or melt) that is delivered through a nozzle (e.g., as a jet ofpolymer solution) and deposited on a target surface. The target surfacecan be the surface of a static plate, a rotating drum (e.g., mandrel),or other form of collector surface that is both electrically conductiveand electrically grounded so that the charged polymer solution is drawntowards the surface.

In some embodiments, the electric field employed is typically on theorder of several kV, and the distance between the nozzle and the targetsurface is usually several cm or more. The solvent of the polymersolution evaporates (at least partially) between leaving the nozzle andreaching the target surface. This results in the deposition of polymerfibers on the surface. Typical fiber diameters range from severalnanometers to several microns. The relative orientation of the fiberscan be affected by the movement of the target surface relative to thenozzle. For example, if the target surface is the surface of a rotatingmandrel, the fibers will align (at least partially) on the surface inthe direction of rotation. In some cases, the nozzle can be scanned backand forth between both ends of a rotating mandrel.

In some embodiments, the size and density of the polymer fibers, theextent of fiber alignment, and other physical characteristics of anelectrospun material can be impacted by factors including, but notlimited to, the nature of the polymer solution, the size of the nozzle,the electrical field, the distance between the nozzle and the targetsurface, the properties of the target surface, the relative movement(e.g., distance and/or speed) between the nozzle and the target surface,and other factors that can affect solvent evaporation and polymerdeposition.

Electrospinning and electrospraying processes may be used for producinginterlinked polymer fiber scaffolds (e.g., hollow synthetic scaffolds)on a mandrel.

Support/Mandrel

In some embodiments, scaffold 10 (e.g., a scaffold having two or morelayers) can be produced using a support (e.g., a solid or hollowsupport) on which the scaffold 10 can be formed. For example, a supportcan be an electrospinning collector, for example a mandrel, or a tube,or any other shaped support. It should be appreciated that the supportcan have any size or shape. However, in some embodiments, the size andshape of the support is designed to produce a scaffold that will supportan artificial tissue of the same or similar size as the gastrointestinaltissue (or portion thereof) being replaced or supplemented in a host. Itshould be appreciated that a mandrel for electrospinning should have aconductive surface. In some embodiments, an electrospinning mandrel ismade of a conductive material (e.g., including one or more metals).However, in some embodiments, an electrospinning mandrel includes aconductive coating (e.g., including one or more metals) covering anon-conductive central support.

It has been found quite unexpectedly that positioning suitable braidmaterial to be integrated in the resulting scaffold 10 at a locationproximate to the surface of the mandrel can serve as an aid tofacilitate removal of the resulting scaffold 10 from contact with themandrel.

Scaffold Properties

It should be appreciated that aspects of the disclosure are useful forenhancing the physical and functional properties of any scaffold, forexample a scaffold based on electrospun and/or electro sprayed fibers.In some embodiments, one or more scaffold components can be thin sheets,cylinders, thick ribs, solid blocks, branched networks, etc., or anycombination thereof having different dimensions. In some embodiments,the dimensions of a complete and/or assembled scaffold are similar oridentical to the dimension of a tissue or organ being replaced. In someembodiments, individual components or layers of a scaffold have smallerdimensions. For example, the thickness of a nanofiber layer can be fromseveral nm to 100 nm, to 1-1000 microns, or even several mm. However, insome embodiments, the dimensions of one or more scaffold components canbe from about 1 mm to 50 cms. However, larger, smaller, or intermediatesized structures may be made as described herein.

In some embodiments, scaffolds are formed as tubular structures that canbe seeded with cells to form tubular tissue regions (e.g., esophageal,or other tubular regions). It should be appreciated that a tubularregion can be a cylinder with a uniform diameter. However, in someembodiments, a tubular region can have any appropriate tubular shape(for example, including portions with different diameters along thelength of the tubular region). A tubular region also can include abranch or a series of branches. In some embodiments, a tubular scaffoldis produced having an opening at one end, both ends, or a plurality ofends (e.g., in the case of a branched scaffold). However, a tubularscaffold may be closed at one, both, or all ends, as aspects of theinvention are not limited in this respect. It also should be appreciatedthat aspects of the invention may be used to produce scaffolds for anytype or organ, including hollow and solid organs, as the invention isnot limited in this respect. In some embodiments, aspects of theinvention are useful to enhance the stability of scaffold or otherstructures that include two or more regions or layers of fibers (e.g.,electrospun nanofibers) that are not physically connected.

In some embodiments, a scaffold is designed to have a porous surfacehaving pores ranging from around 10 nm to about 100 micron in diameterthat can promote cellularization. In some embodiments, pores have anaverage diameter of less than 50 microns, less than 40 microns, lessthan 30 microns, less than 20 microns or less than 10 microns (e.g.,approximately 5, approximately 10, or approximately 15 microns). In someembodiments, pores have an average diameter of 20-40 microns. In someembodiments, pore size is selected to prevent or reduce an immuneresponse or other unwanted host response in the subject. Pore sizes canbe estimated using computational and/or experimental techniques (e.g.,using porosimetry). However, it should be appreciated that pores ofother sizes also can be included.

In some embodiments, a surface layer of a scaffold is synthesized usingfibers that include one or more dissolvable particles that can bedissolved during or after synthesis (e.g., by exposure to a solvent, anaqueous solution, for example, water or a buffer) to leave behind poresthe size of the dissolvable particles. In some embodiments, theparticles are included in the polymer mix that is pumped to the nozzleof an electrospinning device. As a result, the particles are depositedalong with the fibers. In some embodiments, the electrospinningprocedure is configured to deposit thick fibers (e.g., having an averagediameter of several microns, about 10 microns, and thicker). In someembodiments, if the fibers are deposited in a dense pattern, one or morefibers will merge prior to curing to form larger macrostructures (e.g.,10-100 microns thick or more). In some embodiments, thesemacrostructures can entangle two or more layers of fibers and orportions (e.g., fibers) from two or more different components of ascaffold thereby increasing the mechanical integrity of the scaffold. Insome embodiments, when such macrostructures are formed (e.g., viaelectrospinning as described herein) at one or more stages duringscaffold synthesis (e.g., to connect two or more layers and/orcomponents), the surface of the macrostructure(s) can be treated (e.g.,etched or made porous using dissolvable particles as described herein)in order to provide a surface suitable for cellularization.

In some embodiments, the amount of flexible scaffold material (e.g., theslack) between two or more structural components (e.g., rings), betweenstructural members (e.g., arcuate members) of a single continuousstructural component, and/or of a braided support material can be usedto determine the mechanical properties (e.g., tensile strength,elongation, rotation, compression, range of motion, bending, resistance,compliance, degrees of freedom, elasticity, or any other mechanicalproperty, or a combination thereof) of a synthetic scaffold.

In certain embodiments, the scaffold 10 can also include a cellularsheath derived from cells seeded on the outer surface of the scaffoldduring incubation. The cellular sheath adheres to and is in overlyingrelationship to the outer surface of the scaffold. It is contemplatedthat a major portion of the cells present in the cellular sheath will beconnected to the outermost surface of the outer surface and will spanpores defined therein to form a continuous or generally continuoussurface.

In certain embodiments, the cellular sheath can have a thicknesssufficient to provide structural integrity to the sheath layer. Incertain embodiments, the cellular sheath will be composed of a number ofcells which are in contact with the external surface of the scaffoldsufficient to direct regenerating cells in contact with the sheath toproduce a tissue wall that overlays the sheath but does not integratetherewith. In certain embodiments, the sheath can be composed of alining that is between 1 and 100 cells thick on average. Certainembodiments can have a cell thickness between 10 and 100; between 10 and30; between 20 and 30, between 20 and 40; between 20 and 50; between 10and 20; between 30 and 50; between 30 and 60; between 40 and 60; between40 and 70; between 70 and 90.

The scaffold 10 with the associated cellular sheath provides a moveableinsertable device that can be positioned in a suitable gastrointestinalresection site. The scaffold 10 with the associated cellular sheath incontact therewith can be transported to the desired resection site forimplantation. In certain embodiments, the scaffold 10 is configured tobe removable from the implantation site after suitable regeneration ofthe resected organ. In certain embodiments, the removed scaffold willinclude some or all of the cellular sheath connected thereto.

Also disclosed is are various embodiments of method of regenerating atubular organ such as a gastrointestinal organ. In certain embodiments,the method 100 includes the step of resecting a that comprises resectinga portion of a tubular organ in a subject as at reference numeral 110.The organ to be resected can be a tubular organ of the gastrointestinaltract that has been damaged or compromised by disease injury, trauma orcongenital conditions. In certain embodiments, non-limiting examples ofsuitable organs include one of the esophagus, rectum and the like. Incertain embodiments, suitable organs include at least one of theesophagus, small intestines, colon, rectum.

The resection can be achieved by any suitable surgical procedure andproduced a resected organ portion that remains connected to thegastrointestinal tract and remains in the subject after resection. Theresection operation can yield suitable resection edges in certainembodiments.

After resection is completed, a synthetic scaffold is implanted at thesite of the resection as at reference numeral 120. In certainembodiments, implantation can include the step of connecting therespective ends of the resected organ as it remains in the subject torespective ends of the synthetic scaffold such that the syntheticscaffold and at the resected organ can achieve a suitable junctionbetween the respective members. This can be achieved by one or more ofsutures, bioorganic tissue glue, etc.

In certain embodiments, the synthetic scaffold that is implanted can bea tubular member that has an outer polymeric surface and a cellularizedsheath layer overlying at least a portion of the of the outer polymericsurface. Various embodiments of the synthetic scaffold have beendiscussed and can be employed and utilized in the method disclosedherein. In certain embodiments, the synthetic scaffold will include afirst end and a second end opposed to the first end, an outer polymericsurface positioned between the first end and the second end and acellularized sheath layer overlying at least a portion of the outerpolymeric surface. In certain embodiments, the implantation step can beone that brings at least a portion of the cellularized sheath layer intoproximate contact with to at least one of the resection edges of theresected organ portion.

In certain embodiments, the method as disclosed herein also includes thestep of maintaining the synthetic scaffold at the resection site for aperiod of time sufficient to achieve guided tissue growth along thesynthetic scaffold as at reference numeral 130. In certain embodiments,the guided tissue growth is derived from and is in contact with thetissue present in the resected organ portion remaining in the subject.In certain embodiments, the guided tissue growth will be contiguous withthe associated regions of the resected organ. In certain embodiments,the guided tissue growth will exhibit differentiated tissue. In certainembodiments, the guided tissue growth will parallel the outer surface ofthe cellularized sheath layer at a position outward thereof. In certainembodiments, the guided tissue growth is derived from and is in contactwith the tissue present in the resected organ portion remaining in thesubject and will be contiguous with the associated regions of theresected organ. The guided tissue growth will exhibit differentiatedtissue growth and can be parallel the outer surface of the cellularizedsheath layer at a position outward thereof.

After the guided tissue growth has been achieved, the process 100 asdisclosed herein can include step of removing the synthetic scaffold asat reference numeral 140. In certain embodiments, the removing stepoccurs in a manner such that the guided tissue growth remains in thecontact with the resected portion of the organ remaining in the subject.In certain embodiments, the removal process can include intrascopicallyremoving the synthetic scaffold from the interior of the guided tissuegrowth.

In certain embodiments, the synthetic scaffold can be constructed inwhole or in part from bioabsorbable polymeric material. In suchsituations, the method as disclosed herein can include the step ofmaintaining contact between the synthetic scaffold and the resectionedge for an intervals sufficient to achieve guided tissue growth alongthe synthetic scaffold such that at least a portion of the syntheticscaffold is absorbed at the site of resection within a period of timesufficient to achieve guided tissue growth along the synthetic scaffold.In certain embodiments where the scaffold is composed entirely ofbioabsorbale material, the scaffold will be configured to maintainstructural integrity during guided tissue growth. In certainembodiments, where the synthetic scaffold is composed of bioabsorbablematerial in selected regions, it is contemplated that the remainder ofthe scaffold can be removed by suitable procedures after the guidedtissue growth has been achieved.

Guided tissue growth can be monitored by suitable means. In certainembodiments, tissue growth can be monitored endoscopically.

In certain embodiments of the method as disclosed herein, the method canalso include the step of imparting cellular material onto the polymericsurface of the synthetic scaffold and allowing the cellular material togrow to form the cellular sheath layer, the imparting and allowing stepsoccurring prior to the resecting step.

In certain embodiments, the synthetic scaffold that is employed in themethod disclosed herein a tubular member where the outer surfaceincludes spun polymeric fibers. In certain embodiments, the spun fiberscan be electrospun by suitable methods such as those described in thisdisclosure. The cellularized sheath layer spans at least a portionoutwardly positioned electrospun fibers in certain embodiments. Thecellularized sheath layer can is composed of cellular material, thecellular material including at least one of mesenchymal cells, stemcells, pluripotent cells. The cellular material can be autologouslyderived from the subject or can be allogenically derived.

Without being bound to any theory, it is believed that implanting asynthetic scaffold such as those as variously disclosed herein,particularly one seeded with an overlying cellular sheath, promotesgrowth, regeneration and differentiation of the subject tissue incontact with or proximate to the location of the implanted syntheticscaffold. The growing regenerating tissue is guided by the syntheticscaffold and associated sheath to produce a tubular cellular body thatis integrally connected to the resected ends of the remaining tubularorgan and outwardly flaring to encapsulate the synthetic scaffold andassociated cellular sheath layer. It is believed that the scaffold andassociated cellular sheath layer may promote or stimulate regenerativegrowth of the resected tissue while minimizing tissue rejectionresponses. It is also believed that the presence of the cellular sheathlayer can reduce or minimize penetration of the regenerated tissue intothe sheath layer during growth and differentiation. In certainembodiments, tissue generation proceeds from the respective ends towardthe middle. Once the regenerated tissue is in position, the syntheticscaffold can be removed. In certain embodiments, immediately after theremoval of the synthetic scaffold, the regenerated tissue structure willlack the inner epithelial layer. This layer has been seen to regenerateafter removal of the scaffold as illustrated in FIGS. 11A, 11B and 11Ctaken immediately after scaffold removal, 2 months post removal and 3months post removal respectively.

In order to further understand the present disclosure, reference is madeto the following Examples. These Examples are included for purposes ofillustration and are to be considered illustrative of the presentdisclosure and the invention as set forth in the claims.

EXAMPLES Example I Esophageal Scaffolds

Synthetic esophageal scaffolds were produced containing three layers ofmaterial as illustrated in FIG. 1A. A first layer of polyurethane (PU)was deposited onto a metallic mandrel via electrospraying. A braidedmaterial was then deposited on the first PU layer. A second PU layer wasthen deposited via electrospinning. The resulting scaffolds were thenremoved from the mandrel. Each scaffold defined a tubular structurehaving a wall that included three layers (a braided layer sandwichedbetween and inner electro sprayed layer and an outer electrospun layer).Physical dimensions of the scaffold were determined by scanning electronmicroscopy (SEM). The average scaffold wall thickness was approximately500 microns. A non-limiting SEM view of a cross-section of the wall isshown in FIG. 1B. A non-limiting visual image of a cross-section of thetubular scaffold is shown in FIG. 1C. This image shows that thecross-section is approximately “D” shaped. This can be obtained by usinga mandrel that has a “D” shaped cross section.

The outer electrospun layer was a layer of polymer fibers definingpores. The average fiber diameter in the outer layer was approximately3-6 microns The average pore size was approximately 15-20 microns, andthe median pore size was approximately 25-45 microns.

Scaffolds were attached to a support capable of rotating in a bath ofliquid medium within a bioreactor chamber. The rotating mechanism caninclude magnetic drives that allow the support along with the attachedscaffold to be rotated around its longitudinal axis within the liquidbath.

Scaffolds were seeded with cells (e.g., MSCs or other stem cells) bydepositing cell solutions on the external scaffold surface. The seededscaffolds were then incubated in liquid media that supports cell growthby rotating the scaffolds in a bath of the liquid media within abioreactor chamber for approximately one week. The resulting scaffoldsinclude a cellular sheath that is in overlying relationship to the outersurface of the scaffold. In certain embodiments, the cellular sheath canhave a thickness sufficient to provide structural integrity to thesheath layer. In certain embodiments, the cellular sheath will becomposed of a number of cells which are in contact with the externalsurface of the scaffold sufficient to direct regenerating cells incontact with the sheath to produce a tissue wall that overlays thesheath but does not integrate therewith. In certain embodiments, thesheath can be composed of a lining that is between 1 and 100 cells thickon average. Certain embodiments can have a cell thickness between 10 and100; between 10 and 30; between 20 and 30, between 20 and 40; between 20and 50; between 10 and 20; between 30 and 50; between 30 and 60; between40 and 60; between 40 and 70; between 70 and 90.

The scaffold 10 having the seeded cellular sheath can be implanted in tothe resection site and can be positioned in place. It is contemplatedthat the seeded cells present in the sheath can continued to grow postimplantation. In such situations, the seeded cells present in the sheathwill maintain and support a structure that is separate from and tandemto the tissue regenerating at the implantation site.

The respective scaffolds were then implanted into esophageal sites inpigs. An approximately 5 cm section of esophagus was removed andreplaced with a scaffold section that was sutured to the ends of theremaining esophageal tissue in the subject.

The regeneration of esophageal tissue was monitored endoscopically forseveral weeks.

The esophagus is a long muscular tube that has cervical, thoracic, andabdominal parts. FIG. 2 is a diagram that illustrates a cross-section ofan esophagus in a human. In an adult human the esophagus can be 18 cm to25 cm in length. An esophagus wall is composed of striated muscle in theupper part, smooth muscle in the lower part, and a mixture of the two inthe middle. Accordingly, provided herein, in some embodiments, aremultilayered synthetic scaffolds that can promote repair andregeneration of esophageal tissue having two or more layerscorresponding to natural esophageal tissue layers.

FIG. 3 shows stained cross-sections of native and regenerated esophagealtissue 1-2 weeks after an esophageal scaffold implant in a pig. Thecross section shows regeneration of essentially all the esophagealtissue layers (including different muscle and gland layers). Furtheranalysis of the regenerated tissue revealed that the scaffold itself wasnot incorporated into the regenerated esophageal wall. The scaffold wasstill present within the esophagus, but appeared to have acted as aguide that stimulated esophageal regeneration as opposed to becoming anintegral part of the regenerated esophagus.

Example II Esophageal Implant

Synthetic esophogeal scaffolds were produced that contained three layersas illustrated in FIG. 1A with the outer electrospun layer ofpoly-carbonate-polyurethane being deposited as a solution ofpolycarbonate polyurethane dissolved in Hexafluoroisopropanol (HFIP)(DuPont, Wilmington, Del., USA) at 12% w/v. The electrospinningapparatus used was commercially available from IME Technologies,Geldrop, Netherlands. The electrospun fibers were collected on a targetaluminum mandrel rotating at 800 rpm and placed at a distance of 22 mmfrom the syringe tip to deposit an isotropic fiber to produce a scaffoldhaving an average wall thickness of 500 microns. The scaffolds weredried in a vacuum to remove residual solvent. The scaffolds were thenplasma treated with 2 consequent cycles of ethylene and oxygen gasesusing a low pressure plasma system (Diener Tetra 150-LF-PC-D). Scaffoldswere gamma sterilized (STERIS, Northborough, Mass.). The applied doserange was 25-35 KGy.

The resulting tubes were polymeric scaffolds composed of electrospunpolyurethane having a consistent outer diameter (OD) of 22 mm and alength of 11 cm.

The morphology of the electrospun fibers was analyzed by scanningelectron microscopy (Zeiss-EVO MA10). Samples of the scaffolds weresputter coated with Platinum and Palladium using a sputter coater fortwo minutes (Cressington-208HR, TED PELLA, Inc, Redding, Calif.) under apressure of 8×10⁻² mbar and an electric potential of 300 V. Porosity wascalculated using gravimetric measurements. Porosity, ε, is defined interms of the apparent density of the fiber mat, pAPP and bulk density ofthe polymer, ρPU of which it is made: ε=1−ρAPP/ρPU. The apparentscaffold density ρAPP was measured as mass to volume ratio on 10 mm drydisks: ρAPP=Mass/VPU. Pore size measurements were taken using a mercuryporosimeter system (Micromeritics AutoPore IV). Tensile tests on wereperformed consistent with ASTM D638 guidelines on 10 mm×40 mm samplesthat were mounted on an electromechanical load frame (Instron 5943Apparatus) using a 1 kN load cell. The testing parameters were the samefor all samples, at a 100 Hz data acquisition rate, a gauge length of 30mm, and a test speed of 1 mm/sec. Scanning electron microscopy atincreasing magnifications as illustrated in FIG. 7A demonstrated theisotropic fiber arrangement aspects of the electrospun syntheticscaffold. The smooth surface and isotropic nature of the fibers insuresstrength and elasticity of the scaffold is uniform in all directions.

Tensile testing via uniaxial mechanical loading was performed on threepre-implantation and three post-implantation scaffolds (FIG. 7B), whichall showed similar results at in vivo loading values. Consistencybetween the six samples at in vivo loading shows that the scaffolds havea low degree of variability present after fabrication and in vivoimplantation (FIG. 7B, C). The mean (±SD) tensile strain rangedbetween119.5±1.61 mm and 124.5±3.44 mm across the six scaffolds. Atfailure, the tensile strain for the samples pre-implantation reached397.38%±5.52% and post-implantation 408.61%±17.64%. Strain values above400% suggest the reliability of the fabrication process and relative invivo stability. Tensile stress at failure was 7.25±0.59 MPa and4.43±0.77 MPa for pre- and post-implantation scaffolds, respectively.Consequently, the Young's modulus was larger in the pre-implantationsamples than the post-implantation samples, though both groups werecomparable in elasticity at in vivo strains (FIG. 7B, C). The load atfailure followed the same trend as the Young's modulus, with thepre-implantation values being greater than the post-implantation values.

Autologous porcine adipose-derived mesenchymal stem cells (aMSCs) wereisolated from 8 pigs following an open adipose biopsy and analyzed forcharacterization. The 8 Yucatan mini-pigs underwent general anesthesiaand chlorhexidine skin preparation prior to a sterile, open adiposetissue biopsy taken from the lateral abdominal wall. A 5 cm incision wasperformed next to the linea alba with hemostasis achieved usingelectrocautery. Approximately 30-50 g of adipose tissue was isolated,and transferred to a 50mL conical tube containing alpha MinimalEssential Medium (MEM)/glutamax (Thermo Fisher Scientific, Waltham,Mass.) and 1% penicillin/streptomycin (Thermo Fisher Scientific).

20-60 g of abdominal adipose tissue was surgically excised from eachanesthetized Yucatan mini pig (50-60 kg body weight). The tissue sampleswere washed 3 times in alpha Minimal Essential Medium (MEM)/glutamax(Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo FisherScientific). The washed tissue was trimmed to remove lymph nodes andblood vessels and minced into pieces smaller than 5 mm. The tissuepieces were dissociated in digestion buffer (300 IU/mLcollagenase typeII, 0.1% bovine serum albumin (7.5%, fraction V), 1%penicillin/streptomycin, alpha MEM/glutamax) for 55 minutes at 37° C.,5% CO₂. After quenching in complete growth medium (StemXVivo, R&DSystems, Minneapolis, Minn.) and 1% penicillin/streptomycin), the cellswere centrifuged for 15 minutes at 1500 rpm. The cell pellet wasre-suspended in 5 mL of growth medium and filtered through a 70 μmfilter. The cell filtrate was centrifuged for 5 minutes at 1500 rpm. Thecell pellet was re-suspended in 5 mL of growth medium and cells wereplated according to tissue weight (3 g of adipose tissue isolate per T75flask containing 20 mL growth medium).

Cells were washed twice in PBS without calcium or magnesium (ThermoFisher Scientific) and dissociated using TrypLe (Thermo FisherScientific). The dissociation was quenched with growth medium and thecells were centrifuged at 1000 rpm for 5 minutes. The cell pellet wasre-suspended in 1% bovine serum albumin diluted with PBS. Aliquots of 1million cells were incubated in antibody at 4° C. for 30 minutes in thedark (Supplemental Table 1). The labeled cells were washed 3 times inbuffer and secondary antibodies (Life Technologies, Carlsbad, Calif.)were applied as necessary at 4° C. for 30 minutes in the dark. After afurther 3 washes, the cell suspensions were placed into a 96 well platefor flow cytometry (Guava easyCyte HT, EMD Millipore, Billerica, Mass.).Events representative of live cells were gated on forward and sidescatter values, based upon measurements of viability (ViaCount, EMDMillipore). Cell type analysis was performed using fluorescent eventscompensated against unstained and isotype control antibody stainedsamples. Acquired data was exported and analyzed using standalonesoftware (FlowJo version 10, FlowJo, LLC, Ashland, Oreg.).

To assess colony formation, adipose-derived cells were isolated asdescribed, triturated to a single cell suspension and diluted to 10cells/mL of growth medium. 100 μL of the cell suspension was added toeach well of a 96 well plate (Corning, Inc., Corning, N.Y.) and visuallyinspected for cell number the following day. After 5-7 days, colonies ofcells became visible and medium was changed every 3 days until thecolonies contained at least 50 cells. Wells were counted for thepresence of colonies and expressed as a percentage of total wellsanalyzed.

Pluripotency of isolated adipose-derived cells were determined by theirability to undergo adipogenesis and osteogenesis by chemical induction.Cells were plated in 6-well tissue-culture plates, cultured in completegrowth medium, and allowed to grow to 60% or 100% confluency foradipogenic and osteogenic differentiation, respectively. Upon reachingconfluence, medium was changed to either adipogenic or osteogenicdifferentiation medium (CCM007, R&D Systems, Minneapolis, Minn.). Mediumwas changed every 2 days until 14 days in culture. Cells cultured inadipogenic differentiation medium were stained with Oil Red O (AmericanMasterTech, Lodi, Calif.) to identify lipids and cells cultured inosteogenic medium were stained with Alizarin Red (EMD Millipore) forcalcium deposition.

Concentrations of glucose and lactate were measured in conditionedmedium from bioreactors at the time of seeding and 2, 5 and 7 dayspost-seeding (iSTAT, Abbott, Princeton, N.J.).

Cell supernatants were analyzed for the production of porcine cytokinesand growth factors either by multiplex assay on the Luminex 200 platformor by ELISA at the University of Minnesota Cytokine Reference Laboratoryusing commercially available kits and performed according tomanufacturers' directions. A 13-plex porcine-specific bead-set panel(EMD Millipore) was used to determine levels of porcine VEGF, GM-CSF,IL-1RA, IL-6 and IL-8. Values were interpolated from standard curvesgenerated on each plate using BioPlex software (BioRad, Hercules,Calif.) for the Luminex platform, or Microplate Manager software forELISA plates read on a BioRad 550 plate reader. All samples were assayedin duplicate.

Cells were rinsed in PBS and fixed with 10% formalin for 15 minutes atroom temperature. The cells were gently rinsed 3 times in PBS containing0.1% Triton X-100 (PBS-T) and incubated for 1 hour at room temperaturein 10% normal goat serum (Vector) diluted in PBS-T. The rabbitanti-nestin antibody (Biolegend, 1:100) was diluted in 10% normal goatserum and PBS-T and incubated overnight at 4° C. The cells were rinsedtwice in PBS-T and incubated in fluorescent goat anti-rabbit antibody(Alexa Fluor 594, Thermo Fisher Scientific) at room temperature for 1hour. The cells were rinsed twice and counterstained with4′,6-diamidino-2-phenylindole (DAPI).

After 48 hours at 37° C., the cells were washed twice in phosphatebuffered saline containing calcium and magnesium (Thermo FisherScientific) and replaced with fresh growth medium. Thereafter, culturemedium was replaced every 2 days until the flasks were 70%-80%confluent. At passaging, the cells were dissociated (TrypLe, ThermoFisher Scientific), counted (Countess, Thermo Fisher Scientific) andreplated at 200,000 cells per T175 flask. The cells were passaged twiceprior to seeding of scaffolds.

Each 11 cm long scaffold was placed in a bioreactor and seeded with 32million cells (viability >70%, trypan blue dye exclusion, Countess,Thermo Fisher Scientific) in growth medium supplemented with 0.1875%sodium bicarbonate (Thermo Fisher Scientific), MEM eagle (Lonza) and1.19 mg/mL bovine collagen (Organogenesis) in 0.01M hydrochloric acid.The cells were incubated for 5 minutes at 37° C., 5% CO₂ before 200 mLof growth medium was slowly added to the bioreactor. The bioreactor wasincubated for 7-8 days prior to scaffold implantation. Culture media waschanged every 2 days and taken for various assays described below.

The porcine aMSCs were seeded onto a previously characterized scaffoldand subsequently incubated in a bioreactor. Seeded scaffolds were thenimplanted following esophagus resection in Yucatan mini-pigs untilscaffold removal at 3 weeks (FIG. 6) and reproducibly stained positivefor known MSC markers using anti-porcine CD44, CD73, CD90, CD105, andCD146, antibodies and were negative for CD14, CD45, CD106, CD271, andSLA Class II DR. Greater than 95% of the cultured cells stained positivefor nestin and aSMA, indicating stem cell characteristics are maintainedin culture. Pluripotency was determined by chemically inducing theporcine MSC isolates to undergo adipogenesis and osteogenesis,respectively. These aMSCs were routinely expanded and characterized frompassage 1 to 5, and showed consistent phenotypic and functionalcharacteristics.

Porcine aMSCs grown from passage 2 were seeded onto a polymeric scaffoldand incubated in a bioreactor for 7 days (+/−1 day) at 37° C. A numberof cytokines and growth factors were measured using enzyme-linkedimmunosorbent assay (ELISA) to determine if the seeded aMSCs cultured onthe scaffold secrete factors that may assist in angiogenesis andimmunomodulation. Cell secretion of vascular endothelial growth factor(VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin (IL)-6, IL-8, and IL-1RA was detected in conditioned mediumat levels significantly above medium alone (FIG. 4A). However,additional cytokines, TNF-α, IL-1α, IL-1β, INF-γ, IL-10, IL-12, IL-18,platelet-derived growth factor (PDGF), and regulated on activation,normal T expressed and secreted (RANTES), were measured but notdetected.

Punch biopsies of sections of the seeded graft were taken at the end ofthe incubation time at 7 days, to assess cell health and penetrationinto the scaffold. Cellular health was assessed by immunofluorescencestaining using calcein (live cells) and ethidium bromide (dead cells).Cellular penetration of the scaffold was assessed using ethidium bromidefor cell identification. The populations of live cells attached to thescaffold are indicated by the predominance of calcein staining of thebiopsy samples. On cross sections of the scaffold biopsies the majorityof cellular attachment was present at the surface of the scaffold. Whilethere was some evidence of cellular proliferation and ingrowth withinthe scaffold. Metabolic activity of the implant graft during bioreactorincubation was measured every 48 hours for glucose uptake and lactateproduction. Measurements of conditioned medium consistently indicateddecreased glucose and increased lactate levels over time, bothindicators of continued metabolic cell growth. In addition, cellexpansion over 7 days in the bioreactor was quantified by total DNAcontent which increase several fold over the course of bioreactor cellseeding. Further characterization of cell phenotype on the scaffoldfollowing 7 days incubation shows cells continue to express alpha smoothmuscle actin (aSMA) and nestin.

After endotracheal intubation and induction of general anesthesia,animals were placed in a left lateral decubitus position. Hair wasclipped and Chlorhexidine or povidone iodine was used for skinpreparation and the animal was sterilely draped. A standard rightthoracotomy at the level of the 4^(th) intercostal space on each animalwas performed and the thoracic cavity was entered. Single lungventilation was achieved through the use of a double lumen endotrachealtube. A 4-4.5 cm segment of the esophagus, located in the mid thoracicregion (posterior to the right lung hilum, was circumferentiallymobilized and resected to generate a 6 cm defect (tissue retractionproximally and distally). The seeded scaffold (6 cm length) was thenimplanted using polydioxanone (PDS, Ethicon Inc., Somerville, N.J.)absorbable sutures with anastomosis to the proximal and distalesophagus. After the implantation, a commercially available esophagealstent (WallFlex M00516740, Boston Scientific) was inserted under directendoscopic guidance (Storz Video Gastroscope Silver Scope 9.3 MM×110 CM,Tuttlingen, Germany). Stent deployment was performed under endoscopicand surgical visualization. The esophageal stent was fixed in place tothe normal esophageal tissue using absorbable suture, at both theproximal and distal stent flares.

Postoperatively the animals were adjunct supported by gastrostomyfeeding and maintained on a liquid diet through a feeding tube for 2weeks, a mashed diet for a period of 2 more weeks, and then allowed toeat an oral diet of solid food after for the continuation of the study.

At approximately 21 days following the implantation, the scaffolds wereretrieved endoscopically and aMSC impregnated platelet rich plasma (PRP)gel was applied to improve the healing process of the newly formedesophageal conduit. After PRP application, a new fully coveredesophageal stent (WallFlex™, 12 cm long×23 mm outer diameter, BostonScientific Corporation) was placed across the implant zone to preventstricture formation and to maintain anatomy during regeneration. Everytwo weeks the animals underwent sedation and assessment of theesophageal anastomosis and esophageal stent exchange to allow directvisualization and progression of esophageal regeneration. Follow-upobservations were conducted endoscopically (Storz Video GastroscopeSilver Scope 9.3 MM×110 CM, Tuttlingen, Germany).

Regeneration progression was also assessed by endoscopic inspection.Following scaffold removal. the implant zone was visualizedendoscopically at approximately 3-4 week intervals; 2 representativeanimals are shown (FIGS. 10 and 11). At 3-4 weeks post-implantation,regeneration of the mucosal layer was only partially complete. However,the process of esophageal healing continued with time, indicated by theproximal and distal ends of the mucosal layers forming an initial ridgebefore fusion of the 2 layers and complete mucosal regeneration. Theearly reconstitution of the esophageal continuity and integrity and thesubsequent growth of the submucosa from the two opposite edges of theresection have been consistent across all eight animals; 2 animals havebeen maintained to 8 and 9 months post-surgery and have been withoutesophageal stent respectively for 2 and 3 months without evidence ofstricture or stenosis and have had durable oral intake, with noteworthyweight gain.

In order to ascertain histological similarities of the morphologies ofregenerated and native esophogeal tissue. Samples of tissue were excisedfrom a representative pig esophagus at 2.5 months post-implantation, andinclude both the site of surgery and adjacent distal and proximaltissues for histology. (FIG. 13A, dotted box indicates the histologicalanalysis specimens). Representative images of hematoxylin and eosin(FIG. 13B and D) and Masson's trichrome (FIG. 13C and E) stained tissuesections show histologically intact multi-layered esophageal epitheliaand submucosa and normal inner muscular layer morphology.

Representative immunohistochemical analysis from the regenerated regionis depicted in FIG. 14 which depicted histological analysis of tissuefrom pig esophagus at 2.5 months post implantation of a cellularizedscaffold as described herein. FIG. 14A depicts a macrooscopic image ofexcised esophagus (proximal suture to the left). Samples of tissue wereexcised to include the site of surgery, monitored by endoscopy, withadjacent distal and proximal tissues for histology (dotted box). (FIG.14 B-E) Representative images of hematoxylin and eosin (FIG. 14B, D) andMasson's trichrome (FIG. 14C, E) stained tissue sections. Scale bars:A=6 cm, B, C, D and E=200 μm. Representative immunohistochemicalanalysis demonstrates immunoreactivity for Ki67 (FIG. 14F) suggestingcontinued proliferation of mucosal and submucosal cells, CD31 (FIG.14G), CD3e (FIG. 14H), aSMA (FIG. 14I), transgelin/SM22a (FIG. 14J) anda relative absence of striated myosin heavy chain (K) in tissue at thesite of surgery. Scale bars: F-K=200 tm.demonstrates immunoreactivityfor Ki67 (FIG. 14F) at 2.5 months suggests continued proliferation ofmucosal and submucosal cells, CD31 (FIG. 14G), CD3e (FIG. 7H), aSMA(FIG. 14I), transgelin/SM22a (FIG. 14J) and a relative absence ofstriated myosin heavy chain (FIG. 14K) in tissue at the site of surgery.The predominance of aSMA, SM22a, and relative absence of myosin heavychain suggest that smooth muscle proliferation precedes skeletal musclegrowth.

The synthetic matrix seeded with autologously derived mesenchymal cells(aMSCs) resulted in full longitudinal regeneration of the resectedesophagus with minimal mucosal ulcerations or perforations (Table 1).All animal experienced a full 100% of longitudinal regeneration from 2-9weeks after graft removal with 1 out of 6 animals experiencing bothmucosal ulceration or perforation. No animals experienced leaks over thecourse of the study.

TABLE I Longi- tudinal Scaffold regener- Pig Time length ation MucosalContained No (status) Stent (cm) (%) ulceration perforation Leak 1 2weeks No 4.5 100 No No (euthanized) 2 2 weeks No 4.5 100 No No(euthanized) 3 6 weeks Yes 6 100 No No (euthanized) 4 7 weeks Yes 6 100No No (euthanized) 5 9 weeks Yes 6 100 No Yes (euthanized) 6 9 weeks Yes6 100 Yes No (euthanized) 7 7 months Yes 6 (alive) 8 7 months Yes 6(alive)

Example III Other Gastrointestinal Implant

The process as outlined in Examples I and II is implemented replacinggastrointestinal regions localized to the rectum. Results are similar tothe results outlined previously.

Having thus described several embodiments with respect to aspects of theinventions, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of' or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of and “consisting essentially of shallbe closed or semi-closed transitional phrases, respectively, as setforth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

While the disclosure has been described in connection with certainembodiments, it is to be understood that the disclosure is not to belimited to the disclosed embodiments but, on the contrary, is intendedto cover various modifications and equivalent arrangements includedwithin the scope of the appended claims, which scope is to be accordedthe broadest interpretation so as to encompass all such modificationsand equivalent structures as is permitted under the law.

1. A method, comprising the steps of: resecting a portion of a tubularorgan in a subject, the resection step producing a resected organportion, the resected organ portion remaining in the subject; implantinga synthetic scaffold at the site of resection, the synthetic scaffoldincluding an outer polymeric surface and a cellularized sheath layeroverlying at least a portion of the outer polymeric surface; maintainingthe synthetic scaffold at the resection site for a period of timesufficient to achieve guided tissue growth along the synthetic scaffold,the guided tissue growth derived from and in contact with the tissuepresent in the resected organ portion remaining in the subj ect; andafter achieving guided tissue growth, removing the synthetic scaffoldfrom the implantation site, the removing step occurring in a manner suchthat the guided tissue growth remains in the contact with the resectedportion of the tubular organ remaining in the subject.
 2. (canceled) 3.(canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. The method ofclaim 1 wherein the gastrointestinal organ is an esophagus. 8.(canceled)
 9. (canceled)
 10. (canceled)
 11. The synthetic scaffold ofclaim 19 wherein the synthetic scaffold comprises a tubular memberwherein the outwardly oriented surface includes electrospun polymericfibers and wherein the sheath layer spans at least a portion of theoutwardly positioned electrospun fibers.
 12. (canceled)
 13. Theremovable insert device of claim 28 wherein the polymeric body isconfigured as a the tubular gastrointestinal organ.
 14. (canceled) 15.(canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. A syntheticscaffold comprising: a body section, the body section having a first endand a second end opposed to the first end, the body section furtherhaving a least one portion configured as a tubular member, the bodysection comprising an outwardly oriented surface, the outwardly orientedsurface having at least one region composed of spun polymeric fibers,the spun polymeric fibers having an average fiber diameter between 15 nmand 10 microns, at least a portion of the spun polymeric fibersinterlinked to form pores having an average diameter less than 50microns; and at least one sheath layer, the sheath layer composed ofcellular material, the cellular material including at least one ofmesenchymal cells, stem cells and pluripotent cells, the cellularmaterial present in a defined layer the defined layer being between 1and 100 cells thick.
 20. The synthetic scaffold of claim 19 wherein thespun polymeric fibers are electropsun, are interconnected and form anouter layer of the body section and the body section further comprisesat least one inner layer, the inner layer composed of at least one of apolymeric mesh, a polymeric braided support material, a solid polymericmember, an electrospun layer, the outer layer in overlying contact withthe inner layer.
 21. The synthetic scaffold of claim 19 wherein theelectrospun material has an average fiber diameter of 3 to 10micrometers and is composed of at least one of one of the followingpolymeric materials: polyvinylidene fluoride, syndiotactic polystyrene,copolymer of vinylidene fluoride and hexafluoropropylene, polyvinylalcohol, polyvinyl acetate, poly(acrylonitrile), copolymers ofpolyacrylonitrile and acylic acid, copolymers of polyacrylonitrile andmethacrylates, polystyrene, poly(vinyl chloride), copolymer is ofpoly(vinyl chloride), poly(methyl methacrylate), copolymers ofpoly(methyl methacrylate), polyethylene terephthalate, polyurethane. 22.The synthetic scaffold of claim 19 wherein at least one layer is apolymeric material containing polyethylene terephthalate, polyurethane,blends of polyethylene terephthalate and polyurethane.
 23. The syntheticscaffold of claim 20 polymeric braided support material is composed ofat least one of polyethylene terephthalate, polyurethane, nitinol andmixtures thereof.
 24. (canceled)
 25. The synthetic scaffold of claim 19wherein the sheath layer of cellular material overlays the electrospunfibers present on the outer surface such that the cellular material islocated on the outer surface and spans pores defined therein.
 26. Thesynthetic scaffold of claim 19 further comprising at least one hole,indent, protrusion, or a combination thereof defined proximate to atleast one of the first or second ends that is adapted to assist in atleast one of the following: retrieval of the scaffold from a subjectafter tissue regeneration has occurred around the scaffold at the siteof implantation in the subject or implanting the synthetic scaffold in alocation in the body of a subject.
 27. the synthetic scaffold of claim19 wherein the spun polymeric fibers are electropsun, are interconnectedand form an outer layer of the body section, the electrospun fibershaving an average diameter less than 40 microns.
 28. A removablescaffold device comprising: a polymeric body having a first end and asecond end opposed to the first end, the polymeric body further having aleast one portion configured as a tubular member, the polymeric bodycomprising an outwardly oriented surface, the outwardly oriented surfacehaving at least one region composed of electrospun polymeric fibers, theelectrospun polymeric fibers having an average fiber diameter between 15nm and 10 microns, at least a portion of the spun polymeric fibersinterlinked to form pores having an average diameter less than 50microns; and at least one sheath layer, the sheath layer composed ofcellular material derived from seeded cells, the cellular materialincluding mesenchymal cells and stem cells present in a defined layer,the defined layer being between 1 and 100 cells thick.
 29. The removablescaffold device of claim 28 wherein the sheath layer is suspended inoverlying relationship to at least a portion of the outwardly orientedsurface of the polymeric body.
 30. The removable scaffold device ofclaim 29 wherein the cells in the sheath layer has an outer edgeconfigured for proximal contact with at least one body region internalto a patient.
 31. The removable scaffold device of claim 28 wherein thecellular material present in the cellular sheath includes mesenchymalcells, stem cells and wherein the removable scaffold device furtherincludes materials produced by cellular material present in the cellularsheath.
 32. The removable scaffold device of claim 31 wherein thecellular sheath has a thickness of between 10 and 100 cells and whereinthe sheath layer is suspended in overlying relationship to at least aportion of the outwardly oriented surface of the polymeric body.
 33. Theremovable scaffold device of claim 28 wherein the polymeric body has atleast one layer and has a thickness between 100 nm and 1000 microns. 34.The removable scaffold device of claim 33 wherein the electrospunpolymeric fibers of the electrospun material has an average fiberdiameter of 3 to 10 micrometers and is composed of at least one of oneof the following polymeric materials: polyvinylidene fluoride,syndiotactic polystyrene, copolymer of vinylidene fluoride andhexafluoropropylene, polyvinyl alcohol, polyvinyl acetate,poly(acrylonitrile), copolymers of polyacrylonitrile and acylic acid,copolymers of polyacrylonitrile and methacrylates, polystyrene,poly(vinyl chloride), copolymer is of poly(vinyl chloride), poly(methylmethacrylate), copolymers of poly(methyl methacrylate), polyethyleneterephthalate, polyurethane.